The long term goals of this proposal are to understand how the alternative splicing of the Drosophila Down syndrome cell adhesion molecule (Dscam) gene is regulated and to determine the mechanism which Dscam alternative splicing is mutually exclusive. Dscam contains 115 exons, 95 of which are alternatively spliced. The alternative exons are organized into 4 distinct clusters containing 12, 48, 33 and 2 mutually exclusive exons each. Because the exons within each cluster are alternatively spliced in a mutually exclusive manner, it is possible that 38,016 different Dscam isoforms can be expressed. Dscam is therefore the most extensively alternatively spliced gene known to date. The Dscam proteins function as axon guidance receptors that play an important role in neural development and function. In addition, Dscam has also been shown to function as immune receptors that help to defend the organism against pathogens. Current evidence suggests that the identity of the isoforms expressed in individual neurons is critical for the proper wiring of the nervous system and in hemocytes is important for pathogen recognition. Thus understanding the mechanisms regulating Dscam alternative splicing will provide insight into the genetic program that specifies neuronal wiring and pathogen recognition in Drosophila. This proposal is aimed at understanding the mechanisms involved in regulating the alternative splicing of Dscam. First, we will dissect the splicing regulatory program that controls the expression of specific Dscam isoforms. This will involve making a map of the expression pattern of each of the twelve exon 4 variants in the adult brain. Subsequently, we will investigate the role of candidate splicing regulators in controlling splicing of specific exons in individual neurons in the fly. Second, building on an exciting discovery made during the previous funding period, we will determine the requirement and function of competing RNA base- pairing interactions in mutually exclusive splicing of the exon 6 cluster. Finally, we will investigate the mechanism by which proteins ensure the fidelity of exon 6 mutually exclusive splicing. Together, these experiments will provide significant insight into the mechanisms involved in regulating alternative splicing, the mechanisms responsible for mutually exclusive alternative splicing, and the genetic program that determines the specificity of neural wiring and pathogen recognition in Drosophila.